Mesothelin-targeted chimeric antigen receptors and methods of making them
Inventors
Adusumilli, Prasad S. • Sadelain, Michel • Dimitrov, Dimiter S. • Feng, Yang
Assignees
Memorial Sloan Kettering Cancer Center • US Department of Health and Human Services
Publication Number
US-10538588-B2
Publication Date
2020-01-21
Expiration Date
2035-06-05
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Abstract
The presently disclosed subject matter provides for methods and compositions for enhancing the immune response toward cancers and pathogens. It relates to chimeric antigen receptors (CARs) that specifically target human mesothelin, and immunoresponsive cells comprising such CARs. The presently disclosed mesothelin-targeted CARs have enhanced immune-activating properties, including anti-tumor activity.
Core Innovation
The invention provides chimeric antigen receptors (CARs) that specifically target human mesothelin and immunoresponsive cells comprising such CARs to enhance immune responses toward cancers and pathogens. These CARs include an extracellular antigen-binding domain, a transmembrane domain, and an intracellular domain, with the extracellular domain binding human mesothelin with a high affinity in the range of about 1 nM to about 25 nM. The CARs include specific heavy and light chain variable regions, complementarity determining regions (CDRs), linkers, and may be expressed recombinantly, for example from γ-retroviral vectors.
The problem solved by the invention arises from the challenges of treating solid tumors using CAR-expressing T cell therapy, which include tumor-induced immunosuppressive microenvironments, restricted anatomical compartments that impede T cell trafficking, lack of costimulatory ligands, expression of negative regulators of T cell function, and difficulties in selecting tumor-specific antigens that minimize toxicity. Existing CAR T cell therapies show clinical success in hematologic malignancies but face obstacles in solid tumors related to immune evasion and toxicity, including immunogenicity of murine-derived CARs.
The invention addresses these problems by providing mesothelin-targeted CARs with enhanced immune-activating properties, including anti-tumor activity, and features to minimize toxicity and immunogenicity, such as human-derived scFvs. Immunoresponsive cells, particularly T cells, expressing these CARs can recognize human mesothelin with high specificity, effectively lyse tumor cells, secrete cytokines, and persist in vivo, including through targeted delivery methods like intrapleural administration that improves tumor infiltration and therapeutic efficacy.
Claims Coverage
The patent includes 29 claims, with the first claim being independent, covering a chimeric antigen receptor (CAR) comprising a specific single-chain variable fragment (scFv) with defined complementary determining regions (CDRs) that specifically bind human mesothelin. The claims further cover immunoresponsive cells expressing such CARs, nucleic acids encoding the CARs, vectors comprising such nucleic acids, pharmaceutical compositions, kits, and methods for producing such immunoresponsive cells.
Single-chain variable fragment with defined CDRs specific for human mesothelin
The CAR extracellular antigen-binding domain includes an scFv comprising heavy chain variable region CDR1 (SEQ ID NO:11), CDR2 (SEQ ID NO:12), CDR3 (SEQ ID NO:13), and light chain variable region CDR1 (SEQ ID NO:14), CDR2 (SEQ ID NO:15), CDR3 (SEQ ID NO:16).
Human scFv with high affinity and specificity to human mesothelin
The scFv specifically binds human mesothelin with a mesothelin expression level of about 1,000 or more mesothelin binding sites per cell, and with an affinity (Kd) from about 1 nM to about 25 nM.
scFv comprising specific heavy and light chain variable regions
The scFv comprises a heavy chain variable region of amino acids 1-119 of SEQ ID NO:1, and a light chain variable region of amino acids 1-107 of SEQ ID NO:3 or SEQ ID NO:5.
Extracellular domain features
The extracellular antigen-binding domain may include a linker between heavy and light chain variable regions and a leader covalently joined to the N-terminus of the extracellular domain, which can comprise a CD8 polypeptide.
Transmembrane and intracellular domain composition
The transmembrane domain comprises one or more polypeptides selected from CD8, CD28, CD3ζ, 4-1BB, OX40, ICOS, CTLA-4, PD-1, LAG-3, 2B4, BTLA or combinations thereof. The intracellular domain includes CD3ζ and at least one co-stimulatory signaling region such as CD28 or 4-1BB.
Specific CAR embodiments and recombinant expression
Particular CAR embodiments include M28z (CD28 transmembrane and intracellular domains). The CAR is expressed recombinantly, including from γ-retroviral vectors.
Immunoresponsive cells and related compositions
Immunoresponsive cells comprising the CAR include T cells, Natural Killer cells, cytotoxic lymphocytes, and regulatory T cells. Such cells express from about 1 to 4 vector copies of the CAR. Pharmaceutical compositions and kits comprising such cells or nucleic acids encoding the CAR are claimed.
The claims define CARs with a human-derived scFv containing specific CDRs that bind human mesothelin with high affinity and specificity, transmembrane and intracellular domains incorporating co-stimulatory motifs (notably CD28), immunoresponsive cells expressing these CARs, vectors and nucleic acids encoding the CARs, as well as therapeutic compositions, kits, and methods of producing such cells.
Stated Advantages
The mesothelin-targeted CARs have enhanced immune-activating properties including potent anti-tumor activity, with features minimizing CAR-induced toxicity and immunogenicity.
The CARs allow efficient tumor cell lysis and antigen-specific cytokine secretion, promoting T-cell proliferation and survival without requiring tumor co-stimulatory ligands.
Regional administration (e.g., intrapleural) of CAR T cells results in superior efficacy, reduced tumor burden, improved T-cell infiltration, enhanced functional persistence, and systemic tumor control at lower doses than systemic administration.
CD28 co-stimulation within the CAR enhances T-cell effector functions, especially in CD4+ T cells, which can mediate cytotoxicity and sustain proliferation and cytokine production.
The use of human-derived scFvs reduces the risk of immunogenicity compared to murine antibodies.
Documented Applications
Treatment of cancers, including solid tumors such as mesothelioma, lung cancer, pancreatic cancer, ovarian cancer, breast cancer, colon cancer, pleural tumor, glioblastoma, esophageal cancer, gastric cancer, synovial sarcoma, thymic carcinoma, endometrial carcinoma, stomach cancer, cholangiocarcinoma, and metastatic triple-negative breast cancer.
Treatment or prevention of pathogen infections, including viral infections such as cytomegalovirus (CMV), Epstein Barr Virus (EBV), Human Immunodeficiency Virus (HIV), and influenza virus infections.
Enhancing immune responses in immunocompromised subjects.
Use in adoptive T cell therapies with improved tumor infiltration and overcoming tumor-induced immunosuppression.
Administration via systemic injection or regional delivery (e.g., intrapleural), with regional delivery showing improved efficacy in solid tumors.
Treatment or prevention of inflammatory diseases such as pancreatitis and prevention of graft rejection in organ transplant recipients, including through use of immunoinhibitory cells such as regulatory T cells expressing the mesothelin-specific CAR.
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